Introducing oxygen-containing functional groups is a common and convenient method to increase the hydrophilicity of bioelectrodes. In this study, the effect of oxygen-containing functional groups on biofilm was systematically studied to understand how the electron transfer between electrochemically active bacteria (EAB) and bioelectrode was boosted. After electrolysis pretreatment in sulfuric and nitric acid mixture, the oxygen content of the carbon fiber brushes increased from 4.6% to 30.9%. Comparing with the control, the maximum power density increased by 27.7%, while the anode resistance decreased by 21.8%, because charge transfer resistance significantly reduced. The analysis results showed that the content of c-type cytochromes (c-Cyts) in the EAB biofilm was four times higher than that in the control, while the biomass just slightly increased and the bacteria community was similar with that of the control. These findings suggested that the fundamental reason for the enhanced extracellular electron transfer between EAB and electrode was the increased c-Cyts.